Our current research focuses on protein remodeling by molecular chaperones and the role of chaperones in proteolysis. Molecular chaperones are present in all organisms and are highly conserved. They interact with proteins to mediate protein remodeling, folding, assembly, and disassembly without themselves being part of the final complex. Many chaperones are induced by environmental stresses such as heat shock, oxidative stress, and heavy metals, or pathologic conditions, such as inflammation, tissue damage, infection, and genetic diseases involving mutant proteins. They play a critical role during cell stress to prevent the appearance of folding intermediates that lead to irreversibly damaged proteins and assist in the recovery from stress either by refolding and reactivating damaged proteins or by disaggregating and unfolding damaged proteins and delivering them to compartmentalized proteases. We previously discovered that Escherichia coli ClpA, an AAA ATPase and the regulatory component of ClpAP protease, has molecular chaperone activity. This finding demonstrated that Clp ATPases comprise a family of ATP-dependent chaperones. One of the aims of our group is to elucidate the mechanism of action of Clp ATPases, including ClpA and ClpX, and their role in proteolysis in combination with ClpP, the proteolytic component of the ClpAP and ClpXP proteases. More specifically we are currently asking how ClpA and ClpX, recognize substrates. We have found that recognition signals need not be at the end of a substrate to target the substrate for unfolding and degradation by ClpAP or ClpXP. We are addressing whether or not free ends are involved in the process by testing the ability of cyclic proteins with recognition signals to be unfolded and degraded by ClpAP and ClpXP. We are also studying the mechanism of protein remodeling by the DnaK/Hsp70 chaperone system of E. coli. DnaK and its cochaperone, DnaJ interact with hydrophobic peptides and it has been proposed that binding sites become exposed during the protein denaturation that occurs during heat shock. However, there are several known native proteins that are acted upon by DnaJ and DnaK in non-stress conditions. To understand how DnaJ targets specific native proteins for recognition by the DnaK chaperone system, we investigated the interaction of DnaJ and DnaK with RepA, the initiator protein of plasmid P1. DnaJ and DnaK convert inactive RepA dimers into active monomers that bind oriP1 DNA with high affinity. By characterizing RepA deletion mutants, we identified the region of RepA that interacts with DnaJ. A peptide corresponding to this region inhibited the interaction of RepA and DnaJ. In addition, we found that site-directed RepA mutants with alanine substitutions in this region were less efficiently activated for oriP1 DNA binding by DnaJ and DnaK than wild type RepA. Using similar techniques, we identified a different region of RepA that interacts with DnaK. Two mechanisms have been proposed for the targeting of substrates to DnaK by DnaJ. In both, DnaJ initially binds to the substrate through specific DnaJ binding sites. By the first mechanism, the DnaJ and DnaK binding sites on the substrate are one and the same. Binding of DnaK-ATP to the substrate is concurrent with the dissociation of DnaJ from the substrate and the association of DnaJ with DnaK, in a reaction involving DnaJ- and substrate-dependent ATP hydrolysis by DnaK. In the second mechanism, DnaK and DnaJ associate with different sites on the substrate, generating a ternary complex stabilized by substrate-DnaJ, substrate-DnaK, and DnaJ-DnaK interactions following DnaJ- and substrate-dependent ATP hydrolysis by DnaK. Both models suggest that ADP/ATP exchange, stimulated by GrpE, results in conformational changes in DnaK that cause release of the substrate. Our data support the second mechanism in which a native protein recruits the DnaK chaperone system by specifically and separately interacting with DnaJ and DnaK.